Figure 4
From: Characterisation of a natural variant of the γ-butyrolactone signalling receptor
Verification of full-length scbR M145/M600 in S. coelicolor strains LW34 and LW33. A. PCR was carried out using primers ETseq3 and ETS7 to amplify a 752 bp full-length scbRM145/M600 fragment and a 317 bp fragment from the ΔscbR region. The 317 bp PCR product was only found with the LW33/LW34 parental M145ΔscbR mutant. A full-length scbR M145/M600 fragment was amplified from a wt control (M145) and from LW33 and LW34. Obtained PCR products are indicated with arrows on the left; sizes of the marker bands are given on the right. The M145-/M600-type of the gene was confirmed by DNA sequence analysis (data not shown). B. Southern analysis was carried out using Nco I digested genomic DNA of the four strains. A DIG-labelled DNA probe was used to detect the expected 3.2 kb fragment with M145ΔscbR and 2.37 kb fragments with the wt control (M145) and LW33 and LW34. Detected DNA fragments are indicated with arrows on the left; sizes of the marker bands are given on the right. C. PCR was carried out using primers RCseq31 and scbR-M145_c358 (scbR-M600_c358a) to amplify a 2.8 kb scbRM145 (scbRM600)fragment. The latter are specific for the base change between the two scbR variants. A scbRM145 fragment was only obtained with control strain M145 and with LW34, whereas control strain M600 and LW33 showed a scbRM600 specific PCR product. Using water as template did not give any product. Obtained PCR products are labelled with arrows on the left; a 2.5 kb marker band is indicated on the right.