Construction of S. griseus strains expressing atrA from S. coelicolor
Integrating plasmids pL646 and pL644, which constitutively express wild-type AtrA and an Arg 71 to Ala (R71A) mutant, respectively, were transferred by conjugation from Escherichia coli ET12567 (pUB307) (Flett et al. 1997) to S. griseus ATCC 12475 (Vallins and Baumberg 1985) using an established protocol (Kieser et al. 2000). These constructs were based on a pSET152-based integrating expression construct (Tilley 2003) that uses the constitutive ermE*p promoter (Schmitt-John and Engels 1992; Bibb et al. 1994) and the ribosome-binding site of the tuf1 gene (van Wezel et al. 2000). Exconjugants were selected with apramycin (50 μg ml−1) and the resulting strains were designated BH646, BH644 and BH152, respectively. Integration at the attB site in the S. griseus chromosome (Combes et al. 2002) was confirmed initially by a PCR assay using primers that hybridised to sites within attB (5′-CGG TGC GGG TGC CAG GGC) and the backbone of pSET152 (5′-TTC GGC GGC TTC AAG TTC GG), respectively.
Total DNA was prepared as described previously (Kieser et al. 2000) from mycelia grown in YMPD liquid medium (Ohnishi et al. 1999) and cut using BamHI. After agarose gel electrophoresis, the DNA was transferred to a Zeta-Probe GT blotting membrane (BioRad) using a capillary blotting stack (Maniatis et al. 1982), and fixed to the membrane using a UV Stratalinker (Stratagene). The digoxigenin (DIG)-labelled DNA probe for the atrA gene was generated using the PCR DIG Probe Synthesis Kit (Roche). The primers were AtraAF (5′-GGA TCC TCA GGA TTC TCA TTG GTC GTC) and AtraAR (5′-GGA TCC GTG GCT ATG ACC AGC AGC AC). The membrane was prehybridised at 55°C for at least 30 min using the DIG Easy Hyb hybridisation buffer (Roche), denatured DIG-labelled probe was added and incubated continued overnight at the same temperature. After hybridisation, the membrane was washed twice with low stringency buffer (2 × SSC, 0.1% w/v SDS) at room temperature and twice with high stringency buffer (0.1 × SSC, 0.1% w/v SDS) at 68°C. At this temperature, only genes that have 80–100% sequence identity with the probe are bound (DIG Application Guide for Filter Hybridization, Roche). For the detection of probe, the membrane was washed and blocked using the DIG Wash and Block Buffer Set (Roche), incubated with anti-DIG-alkaline phosphatase antibody (Roche), and then incubated with a chromogenic substrate for alkaline phosphatase, Nitro-Blue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt.
Bioassay of streptomycin
The bioassay of streptomycin essentially followed a method described previously (Horinouchi et al. 1984). The indicator was Bacillus subtilis 168 obtained from Dr. Keith Stephenson, University of Leeds. The specificity was checked by the parallel use of streptomycin-sensitive or -resistant E. coli. 245 × 245 mm2 bioassay dishes (Corning) containing 150 ml of Bennett’s medium in 2% (w/v) agar No. 1 (LAB M) were spotted at 3 cm intervals with 3 × 106 spores. The plates were incubated at 30°C for 2 days before being overlaid with 50 ml of Bennett’s soft agar containing 2.1 ml of a culture of the indicator at OD600 0.5, which had been grown in LB medium at 37°C. After another day of incubation at 30°C, the area of the ring of inhibition of indicator growth around each patch of S. griseus was measured. Standard conditions were used for the preparation and quantitation of spores (Kieser et al. 2000).
Analysis of transcript levels
RNA was isolated, quantitated, and reverse transcribed as described previously (Uguru et al. 2005). The RNA was isolated from S. griseus mycelia scraped from cellophane (AA Packaging, UK) laid on the surface of YMPD agar plates. The level of the strR transcript in samples from mycelial patches of different age was analysed in real time as described previously (Uguru et al. 2005) using primers strR_FOR (5′-GAG CAA GTC CGT GAG AGG TC) and strR_REV (5′-GAG GGA AGC AAT GAT TCG AC). As before (Uguru et al. 2005), the values were normalised using the level of hrdB mRNA as a control. The PCR reaction conditions were 95°C for 7 min followed by 45 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 40 s. The temperature at which fluorescence was measured at the end of each cycle was 83°C. The relative levels of several transcripts in samples from mycelial patches of the same age were analysed semi-quantitatively using end-point analysis. The primers for the amplification of segments of hrdB (127 bp), strB1 (123 bp), strD (125 bp) and adpA (130 bp) mRNA were 5′-TGG TCG AGG TCA TCA ACA AG with 5′-TGG ACC TCG ATG ACC TTC TC, 5′-GAC GGG TTC CAC GAC TAC TG with 5′-CAG CAG GAG GTC CTT GTA G, 5′-GTC GCC GAG ATA CAT GAT GA with 5′-ATG GTT CGA GAT TCG GAC TG, and 5′-CGA TCT CTG CCT CCA CAT AG with 5′-CTC CGG TAA AGA CCT GTC CA, respectively. The size of each amplicon is provided in the parentheses. The PCR conditions for end-product analysis were standard PCR reactions using PCR reaction buffer Mix J (Epicenter) and 10% (v/v) DMSO (Sigma). The reactions were done in 0.2 ml thin-wall tubes (BIOplastics) using a standard PCR machine with a heated lid (MiniCycler, MJ Research) and the final products were analysed by gel electrophoresis in TBE-buffered 2% (w/v) agarose. The reactions were incubated at 95°C for 5 min followed by 40 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 40 s.