Faster Recombinant DNA Procedures for Streptomyces

The great industrial importance of the streptomycetes has stimulated their genetic manipulation (1). The plasmid or phage vectors that have been developed require recombinant selection and screening procedures, which in Streptomyces can take several days and necessitate comparatively large volumes of cultures and reagents (4). To improve the situation, we have coupled a scaled-down procedure for DNA isolation with polymerase chain reaction (PCR) amplification. The maintenance of Streptomyces, S. coelicolor J1501 and S. lividans 1326 and the DNA manipulations and transformation methods were those described previously (4), except where stated otherwise. The microorganisms were grown either on R2YE solid medium (4) or in 20-mL “Universal” bottles containing 5 mL of YEME liquid medium (4) and 3 cm of a stainless steel spring (material type 316; The Alliance Spring Co. Ltd., London, England, UK). Plasmid pIJ486 was used as a vector (3,5). For PCR amplifications, 4-day-old transformed colonies from solid media were picked with a scalpel and crumbled either with a toothpick or by vortex mixing with a small amount of glass beads (0.15-mm diameter) in 500-μL tubes containing 50 μL of TE (10 mM Tris-HCl, 1 mM sodiumEDTA, pH 8.0) (2). Alternatively, up to 5 μL of mycelium harvested from liquid media were used. Following this step, TE and lysozyme (Sigma, St. Louis, MO, USA) were added to a final vol of 100 μL (final concentration of lysozyme 3 mg/mL) and incubated at 37°C for 30 min. Finally, the tubes were placed in boiling water for 15 min, spun in a microcentrifuge at 10000× g for 5 min, and the supernatant (here termed “DNA samples”) was saved for PCR or protoplast transformations. Boiling the samples prevents nuclease activity and spore and/or mycelium cross-contamination in transformation experiments with different hosts. The primary PCR mixture (90 μL) contained 55.5 μL of double-distilled (dd)H2O, 2 μL 5 mM dNTPs, 1 + 1 μL of selected primers (stock solution of each primer at 20 μM), 20 μL 5× Buffer Q (Qiagen Ltd., West Sussex, England, UK), 10 μL 10× PCR Buffer (Qiagen) and 0.5 μL (2.5 U) Taq DNA Polymerase (Qiagen). Each amplification reaction contained 2 μL of DNA sample and 18 μL of primary PCR mixture. In a Model 9600 GeneAmp PCR System (PE Biosystems, Foster City, CA, USA) the amplification consisted of an initial 3-min denaturation step at 94°C and 32 cycles of 1 min at 94°C, 1 min at 58°C and 1 min 30 s at 68°C. The final extension step was 10 min at 68°C. To detect the presence or absence of inserts in pIJ486 after transformation, two primers were designed, A1 (5′-CGCAATTCCTTTAGTTGTTCC-3′) and A2 (5′-GCGAAACGATCCTCATCC3′). Primers A1 and A2 anneal just upstream of the vector’s polylinker [in the disrupted gene melC2 (2)] and a few base pairs downstream of the BclI restriction site of this polylinker, respectively. In the absence of an insert, PCR using both primers amplified a 224-bp fragment, which includes the polylinker of pIJ486 (Figure 1a, lane 2). No cross-amplification with the chromosomal DNA of the host could be observed (Figure 1a, lane 3). Other lanes of Figure 1a show the results using DNA samples from strains transformed with pIJ486 digested with EcoRI and HindIII (Promega UK, Southampton, England, UK) and ligated (T4 DNA ligase; Promega UK) with EcoRI-HindIII DNA fragments of different sizes. In our laboratory, DNA fragments of up to 7 kb were amplified successfully. Upon subsequent transformation of S. coelicolor J1501 with pIJ486 DNA as a control and with DNA samples (2 μL) from two S. lividans transformants (Figure 1a, lanes 5 and 13) using the “quick transformation method” (3), several hundreds of colonies were observed in each transformation. New DNA samples from S. coelicolor transformants were prepared and checked by PCR for the presence of the expected amplified fragments. In all colonies tested we detected the presence of the correct constructs (Figure 1b, lanes 4–6). The same methods proved suitable for the amplification of single-copy chromosomal DNA sequences. Thus, primers internal to single-copy gene wblS, recently sequenced in S. coelicolor (unpublished results) were designed (primer B1, 5′-GGCGTTCCTGAGTTCCAC-3′ and B2, 5′-TAGACCCGTGCTGCAACC-3′). As shown in Figure 1b, two PCR controls performed with isolated genomic DNA and a DNA sample from a strain transformed with pIJ486 gave the same predicted amplification of 349 bp (Figure 1b, lanes 2