PCR was performed using Biomix (Bioline, London, UK) polymerase or HotStar HiFidelity polymerase kit (Qiagen, Crawley, UK) according to the manufacturer’s instructions with the addition of 5% DMSO. Generation of a GlnR deletion mutant and the GlnR D48A point mutation strain were performed using the recombineering method (van Kessel & Hatfull, 2007, 2008a, b). For generation of the GlnR deletion mutant, upstream and downstream sequences flanking glnR (msmeg_5784) were amplified from M. smegmatis genomic DNA by PCR as stated; primer sequences are listed in Table 2. The flanking regions were designed so as not to disrupt any neighbouring

OSI-744 datasheet genes or introduce any downstream effects. Vector pYUB854 was used to subclone the homologous flanking sequences either side of

a hygromycin resistance (HygR) cassette (Bardarov et al., 2002). Allelic exchange sequence (AES) DNA was prepared by digesting the pYUB854_glnR construct with AflII and SpeI. Linear AES DNA (200 ng) was used to transform M. smegmatis cells containing the pJV126 recombineering plasmid (a gift from Graham Hatfull). Putative null mutants were selected on 7H11 Selleck BTK inhibitor agar containing hygromycin (50 μg mL−1) and kanamycin (50 μg mL−1). The GlnR_D48A point mutation was generated using M. smegmatis containing the pJV128 recombineering plasmid (a gift from Graham Hatfull). Cells were cotransformed with 100 ng of two ssDNA oligonucleotides: GlnR_Point_mut containing the base pair changes for the required glnR D48A point mutation and HygS_Repair containing the required base pair changes to convert the hygromycin resistance cassette from nonfunctional to functional (Table 2). Cediranib (AZD2171) This hygromycin resistance repair method was used to select colonies that had undergone recombination. A mismatch amplification

mutation assay (MAMA) PCR screen using primer pairs MAMA_PCR_F and MAMA_PCR_R was performed to identify glnR containing the desired point mutation (Cha et al., 1992; Swaminathan et al., 2001). MAMA PCR conditions were: 95 °C for 5 min, 39 cycles of 95 °C for 15 s, 32 °C for 1 min, with final extension time of 72 °C for 7 min. Recombineering plasmids were removed from both mutant strains via negative sacB selection (Pelicic et al., 1996). Confirmation of a GlnR D48A point mutation was carried out by amplifying the entire glnR genomic region using primer pairs GlnR_reg_F and GlnR_reg_R with high fidelity polymerase, and sequencing the glnR gene with GlnR_D48A_SeqF and GlnR_D48A_SeqR (Table 2). Confirmation of GlnR deletion was carried out by PCR using primers outside the upstream and downstream flanking regions in combination with hygromycin cassette–specific primers (Table 2). PCR products would only be obtained with insertion of the hygromycin cassette by recombination onto the chromosome at the correct location. Further confirmation of GlnR deletion phenotype was provided by Western analysis using a custom-made GlnR polyclonal antibody (Eurogentec, Seraing, Belgium).