A control reaction lacking reverse transcriptase was performed to ensure any resulting amplification in later steps was not the result of contaminating chromosomal DNA.

After A tailing the 3′ end of the cDNA with terminal deoxynucleotide transferase, a second gene specific primer (LK738, see Additional file 5: Table S2) was used to amplify the cDNA (in conjunction with a kit primer). The resulting amplicons were cloned into the pCR™-Blunt vector (Invitrogen) and sequenced using standard M13F and M13R primers. Cloning, expression, and purification of MsvR The MaMsvR gene was PCR amplified with the primers LK588 and 589 (see mTOR inhibitor Additional file 5: Table S2) containing a 5′ BamHI site and a 3′ PstI site, respectively, and cloned into an the pQE80L expression vector (Qiagen) modified with an N-terminal Strep-Tag®. The resulting

plasmid was named pLK314 and transformed into E.coli Rosetta™ (Novagen) for expression. Cells were grown to an OD600 of 0.4 at 37˚C and then induced with 0.1 mM IPTG at 18˚C for 16 hours. Cells were lysed by sonication and the protein was purified with Streptactin resin (Qiagen) according to manufacturer’s recommendation. Reducing SDS-PAGE was employed to ensure no other proteins were present in MsvR preparations. Purified protein was dialyzed into a protein storage buffer (20 mM Tris pH 8, 10 mM MgCl2, 200 mM KCl, 25% glycerol) this website and stored at -20˚C. Protein concentrations were determined by the Bradford assay [38]. MaMsvR was diluted in the same protein storage buffer containing 50% glycerol to 2 μM for use in assays. MaMsvR was treated with 5 mM dithiothreitol (DTT) in reducing reactions. In non-reducing reactions, the protein samples were left untreated after aerobic purification. MthMsvR was purified and treated as previously described [9]. SDS-PAGE gels of representative purifications are shown in (see Additional file 6: Figure S4). MsvR V4R domain cysteine to alanine variants Cysteine codons (TGT) were converted to alanine codons (GCT) using the QuikChange® site click here directed mutagenesis kit (Agilent Technologies). The PAK5 sequence of primers used to generate individual alanine codon substitutions in pLK314

can be found in (see Additional file 5: Table S2). Plasmids resulting from QuikChange® reactions were confirmed by sequencing. The resulting MsvR variants were overexpressed and purified in the same manner as native MsvR. Electrophoretic mobility shift assay (EMSA) Larger DNA templates for EMSA were PCR amplified from M. acetivorans C2A genomic DNA with custom primers (see Additional file 5: Table S2). With the exception of rpoK (MA0599) which is a portion of the open reading frame, all other templates (designated P xxxx ) contain the extreme 5′ end of the predicted open reading frame and ~ 200 bp upstream of the translational start site. All templates were agarose gel purified, purified using the Wizard® SV PCR Clean-Up System (Promega), and confirmed by sequencing.