Using Big Dye?Terminator V3.1 Cycle Sequencing kit (Perkin Elmer).Construction of an amtR deletion strainBacteria were routinely grown at 37 in baffled flasks under agitation. Mycobacterial strains were grown in Middlebrook 7H9 liquid medium (Difco Laboratories; per 900 ml approx. 0.5 g ammonium sulfate, 0.5 g L-glutamic acid, 0.1 g sodium citrate, 1.0 mg pyridoxine, 0.5 mg biotin,To inactivate the AmtR function, a mutant with in-frame deletion of the DNA fragment (amtRDBD) encoding AmtR DNA-binding domain (DBD) (amino acid residue PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28827318 26 to 72) was generated as described earlier [11]. Briefly, two DNA fragments (1 kb up- and downstream of amtRDBD) were amplified via PCR using AG-221 chemical information chromosomal DNA from M. smegmatis strain SMR5 as a template. SwaI and PacI restriction sites were introduced into the primer sequences used for amplification of the upstream arm and SpeI and PmeI sites into the primer sequences used for amplification of the downstream arm (Additional file 1: Table S3). The PCR products were then cloned in vector pML814 (ColE1 origin, FRT-hyg-FRT, rpsL, AmpR, HygR, 6220 bp, general deletion vector), resulting in the recombinant plasmid pML814amtRDBD, which carried a FRThyg-FRT expression cassette [34] flanked by the upstreamJe erger et al. BMC Research Notes 2013, 6:482 http://www.biomedcentral.com/1756-0500/6/Page 16 ofand downstream arms. M. smegmatis SMR5 was transformed with pML814amtRDBD and the transformants were first selected on hygromycin-containing plates to obtain single crossovers [34]. After verification of the single crossover event via colony PCR, cells were further selected on hygromycin/streptomycin containing plates. Clones on these plates should have lost the vector and have the integrated FRT-hyg-FRT cassette in the chromosome. After verification of the double crossover event via PCR, the FLP recombinase was used to specifically remove the hyg gene from the chromosome, thus generating a marker-free deletion mutant. Selection of clones was performed using hygromycin/streptomycin-containing plates. Deletion of amtRDBD in the resulting strain MH2 was verified by PCR and Southern blotting (data not shown). On basis of the mutant strain YL1, glnR was further deleted as described earlier [11], generating the mutant strain YL2, with the inactivation of both amtRDBD and glnR, which was confirmed by PCR and Southern blotting (data not shown).Construction of amtR carrying plasmidsPCR and subsequent labelling with DIG RNA-labelling mix (Roche, Mannheim) and T7 polymerase (NEB, Frankfurt). RNA (1 g per time point) was spotted onto nylon membranes using a Schleicher Schuell (Dassel) Minifold I Dot Blotter. Hybridization of digoxigeninlabelled RNA probes was detected with X-ray films (Amersham Hyperfilm MP; GE Healthcare) using alkaline phosphatase-conjugated anti-digoxigenin Fab fragments and CSPD as light-emitting substrate as recommended by the supplier (Roche, Mannheim). All experiments were carried out at least twice with independent cultures (biological replicates).Real-time reverse transcriptase PCRFor complementation assays, the amtR gene was amplified by PCR using chromosomal DNA of M. smegmatis as template and the primers amtRcom-fw/amtRcom-rev (see Additional file 1: Table S3). The PCR products were ligated to plasmid pMN016 (psmyc-mspA, ColE1 origin, pAL5000 origin, HygR, 6164 bp, [35]) between the SwaI and PacI sites. For the overexpression of AmtR protein in E. coli, the amtR gene was amplified by PCR (for prime.