Construction of GusA reporter plasmids and β-glucuronidase activity measurements

Plasmids padpAscript, pmoeE5script and pbldAscript for the study of transcriptional activity of adpA_gh, moeE5 and bldA_gh promoters, respectively, were constructed previously (23).

To probe the activity of rmdB_gh promoter, a DNA fragment comprising 0.4 kb at the front of the translation start codon was amplified by PCR with appropriate primers. The rmdB_ghp fragment was cloned into XbaI/KpnI-linearized pGUS to give prmdBscript. To evaluate the expression of rmdB_gh on the translational level, a DNA region containing the entire stop codon-free gene along with putative promoter (400 bp upstream of the translation start codons) was amplified by PCR using an appropriate pair of primers. The amplicon was ligated to XbaI/EcoRV-cleaved pGUSHL4aadA, an integrative Streptomyces vector where the examined gene is fused to the gusA reporter gene through the helical peptide linker HL4, yielding prmdBtransl.

In order to substitute the TTA codon to CTG, PCR mutagenesis was applied to amplify an 8.2 kb DNA fragment from pSETrmdB with appropriate primers carrying a single codon TTA→CTG substitution. The obtained amplicon was treated with T4 Polynucleotide kinase and then self-ligated giving pSETrmdB-CTG. Next, a 2.7 kb DNA region comprising stop codon free rmdB_gh along with its putative promoter region was PCR amplified and cloned into pGUSHL4aadA using the aforementioned procedure to create prmdB-CTGtransl. In control experiments, promoter free rmdB_gh(TTA) and rmdB_gh(CTG) genes without stop codon were amplified by PCR using respective set of primers and cloned into XbaI/EcoRV-digested pGUSHL4aadA, resulting in prmdBcontr and prmdB-CTGcontrol, respectively.

Measurement of β-glucuronidase activity was carried out as described previously (23). Cultures and subsequent β-glucuronidase assays were performed in triplicate. Values were normalized to equal amounts of dry biomass (10 mg) and presented as the mean ±2 standard deviations.